EP2720041B1 - Améliorant d'agglutination - Google Patents

Améliorant d'agglutination Download PDF

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Publication number
EP2720041B1
EP2720041B1 EP12796593.7A EP12796593A EP2720041B1 EP 2720041 B1 EP2720041 B1 EP 2720041B1 EP 12796593 A EP12796593 A EP 12796593A EP 2720041 B1 EP2720041 B1 EP 2720041B1
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Prior art keywords
general formula
polymer
agglutination
immunoagglutination
reagent
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German (de)
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EP2720041A4 (fr
EP2720041A1 (fr
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Naoyuki Yamamoto
Tsutomu Masuda
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Fujifilm Wako Pure Chemical Corp
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Wako Pure Chemical Industries Ltd
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/5306—Improving reaction conditions, e.g. reduction of non-specific binding, promotion of specific binding
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
    • G01N33/542—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with steric inhibition or signal modification, e.g. fluorescent quenching
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
    • C08F20/00—Homopolymers and copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and only one being terminated by only one carboxyl radical or a salt, anhydride, ester, amide, imide or nitrile thereof
    • C08F20/02—Monocarboxylic acids having less than ten carbon atoms, Derivatives thereof
    • C08F20/10—Esters
    • C08F20/34—Esters containing nitrogen, e.g. N,N-dimethylaminoethyl (meth)acrylate
    • C08F20/36—Esters containing nitrogen, e.g. N,N-dimethylaminoethyl (meth)acrylate containing oxygen in addition to the carboxy oxygen, e.g. 2-N-morpholinoethyl (meth)acrylate or 2-isocyanatoethyl (meth)acrylate
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
    • C08F220/00—Copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and only one being terminated by only one carboxyl radical or a salt, anhydride ester, amide, imide or nitrile thereof
    • C08F220/02—Monocarboxylic acids having less than ten carbon atoms; Derivatives thereof
    • C08F220/10—Esters
    • C08F220/34—Esters containing nitrogen, e.g. N,N-dimethylaminoethyl (meth)acrylate
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor

Definitions

  • the present invention relates to an agglutination enhancer for use in an immunoagglutination measurement method, and the immunoagglutination measurement method using the agglutination enhancer.
  • an immunoagglutination measurement method it has been carried out conventionally as an immunoagglutination measurement method that the presence of an analyte is confirmed or the concentration of an analyte is measured from the degree of the agglutination generated with the use of an insoluble carrier such as a latex immobilized an antigen or an antibody reacting with the analyte, in order to determine the presence or the concentration of the analyte in a biological sample such as serum, plasma, and urine.
  • an insoluble carrier such as a latex immobilized an antigen or an antibody reacting with the analyte
  • an immunoagglutination enhancer which facilitate the generation of agglutination based on an antigen-antibody reaction is usually employed for the purpose of improving the sensitivity.
  • an immunoagglutination enhancer polyethylene glycol (PEG) and the like have been known well, however, the PEG has a problem such that the PEG salts out in a solution with high salt concentration, causing a blank value increased, and therefore accuracy of determination becomes worse.
  • Patent Literature 1 a polymer containing methacryloyloxyethyl phosphorylcholine (MPC) as an immunoagglutination enhancer which solves such problem has been devised.
  • MPC methacryloyloxyethyl phosphorylcholine
  • Patent Literature 1 JP A-2002-365296 .
  • the present invention is to provide an agglutination enhancer which enables high sensitive immunoagglutination measurement, and which shows a superior agglutination enhancing effect to those of the conventional immunoagglutination enhancers.
  • a polymer having monomer units shown by the following general formula [1] (Wherein R 1 represents a hydrogen atom or a methyl group, R 2 and R 3 represent independently a methyl group or an ethyl group, respectively, X represents -NH-or an oxygen atom, n represents an integer of 1 to 6, and m represents an integer of 1 to 3) shows superior agglutination enhancing effect to those of conventional immunoagglutination enhancers, and have thus completed the present invention.
  • the present invention relates to the use of "an agglutination enhancer for immunoagglutination measurement method comprising a polymer having a monomer unit shown by the following general formula [1] (Wherein R 1 , R 2 , R 3 , X, n and m are the same as described above.), a reagent for immunoagglutination measurement method comprising an agglutination enhancer in which the above-described polymer is a copolymer having a monomer unit shown by the above-described general formula [1] and a monomer unit shown by the following general formula [2]
  • R 4 represents a hydrogen atom or a methyl group, and k represents an integer of 1 to 10.
  • an immunoagglutination measurement method which comprises that in the coexistence of the above-described agglutination enhancer for immunoagglutination measurement method, an antibody against the analyte or an antigen for the analyte is brought into contact with the analyte to cause an antigen-antibody reaction.
  • the immunoagglutination enhancer of the present invention has a greater agglutination enhancing effect as compared with the conventional immunoagglutination enhancers. Therefore, generation of agglutination by an antigen-antibody reaction is made easy by employing immunoagglutination enhancer of the present invention, in the immunoagglutination measurement method such as turbidimetric immunoassay and nephelometric immunoassay which utilize the agglutination of antigen-antibody reaction product produced with the use of a carrier such as latex particles to which antigen or antibody has been immobilized. As a result, the immunoagglutination enhancer of the present invention enables a highly sensitive measurement.
  • the polymer to be used in the agglutination enhancer for immunoagglutination measurement method of the present invention may be either homopolymer or copolymer as long as it has a monomer unit shown by the general formula [1].
  • said copolymer includes, for example, the one which consists of a monomer unit shown by the general formula [1] and a monomer unit shown by the general formula [2].
  • the weight-average molecular weight of the polymer to be employed in the agglutination enhancer of the present invention is usually 50,000 to 3,000,000, preferably 100,000 to 3,000,000, and more preferably 200,000 to 3,000,000.
  • its weight-average molecular weight is usually 50,000 to 3,000,000, preferably 100,000 to 3,000,000, and more preferably 200,000 to 3,000,000.
  • a specific preferable example of the monomer unit shown by the general formula [1] includes, for example, the following general formula [1-1] to [1-8].
  • the general formulae [1-1] to [1-4] are preferable, the general formula [1-1], the general formula [1-3], and the general formula [1-4] are more preferable, and the general formula [1-1] is particularly preferable. More specifically, the ones which are shown by the following [1-1-1], [1-1-2], [1-3-1], and [1-4-1] are preferable:
  • the homopolymer which has monomer unit shown by the general formula [1] can be produced by reacting, for example, a (meth) acrylic acid derivative shown by the following general formula [3]
  • R 1 , R 2 , R 3 , X, and n are the same as described above
  • a specific example of the (meth) acrylic acid derivative shown by the general formula [3] includes, for example, N-[2-(dimethylamino) ethyl] methacrylate, N-[3-(dimethylamino) propyl] methacrylate, N-[4-(dimethylamino) butyl] methacrylate, N-[5-(dimethylamino) pentyl] methacrylate, N-[6-(dimethylamino) hexyl] methacrylate; N-[2-(dimethylamino) ethyl] acrylate, N-[3-(dimethylamino) propyl] acrylate, N-[4-(dimethylamino) butyl] acrylate, N-[5-(dimethylamino) pentyl] acrylate, N-[6-(dimethylamino) hexyl] acrylate; N-[2-(d
  • N-[2-(dimethylamino) ethyl] methacrylate, N-[4-(dimethylamino) butyl] methacrylate, N-[3-(dimethylamino) propyl] acrylamide, N-[3-(dimethylamino) propyl] methacrylamide, etc. are particularly preferable.
  • (meth) acrylic acid derivatives commercially available products or the one synthesized appropriately from (meth) acrylic acid by a conventional method may be employed.
  • a halogen atom represented by X in the general formula [4] includes, for example, fluorine, chlorine, bromine, iodine etc., and, chlorine and bromine are particularly preferable.
  • a specific example of carboxylic acid compound shown by the general formula [4] includes, for example, chloroacetic acid, fluoroacetic acid, bromoacetic acid, iodoacetic acid, chloropropionic acid, fluoropropionic acid, bromopropionic acid, iodopropionic acid, chlorobutanoic acid, fluorobutanoic acid, bromobutanoic acid, iodobutanoic acid etc., and, bromoacetic acid, chloropropionic acid, and chloroacetic acid are preferable, and chloroacetic acid is particularly preferable among them.
  • carboxylic acid compound shown by the general formula [4] may be usually 0.5 to 3 times mole, preferably 1 to 2 times mole of the (meth) acrylic acid derivative shown by the general formula [3].
  • a reaction solvent to be used at the time of a reaction of the (meth) acrylic acid derivative shown by the above-described general formula [3] with the carboxylic acid compound shown by the general formula [4] or the salt thereof includes, for example, hydrocarbons such as toluene, xylene, benzene, cyclohexane, n-hexane, and n-octane, for example, alcohols such as methanol, ethanol, n-propanol, isopropanol, n-butanol, isobutanol, and tert-butanol, dimethylformamide (DMF) and water etc., and, the alcohols are preferable, and among them, methanol, ethanol, n-propanol and isopropanol are preferable.
  • hydrocarbons such as toluene, xylene, benzene, cyclohexane, n-hexane, and n-o
  • reaction solvent may be used by mixing two or more kinds appropriately.
  • the amount of reaction solvent to be used is usually 100 to 300 mL for 50 g of the total amount of (meth) acrylic acid derivative shown by the general formula [3] and carboxylic acid compound shown by the general formula [4] or the salt thereof.
  • the temperature of the reaction of the (meth) acrylic acid derivative shown by the general formula [3] with the carboxylic acid compound shown by the general formula [4] or the salt thereof may be set appropriately according to the reaction solvent, etc., and, usually it is 20 to 120°C, preferably 40 to 80°C, and the reaction time is usually 1 to 20 hours, preferably 5 to 12 hours.
  • the polymerization reaction in the method for production of the polymer which has a monomer unit shown by the general formula [1] can be performed by a method well known per se such as, for example, solution polymerization, mass polymerization, emulsification polymerization, and suspension polymerization.
  • polymerization initiator for example, azo compounds such as azoisobutyronitrile, 2, 2'-azobis (2, 4-dimethylvaleronitril), 2, 2'-azobis (methyl 2-methylpropionate), 2, 2'-azobis (2-methylbutyronitril), peroxides such as benzoyl peroxide, lauroyl peroxide, potassium peroxodisulfate, and ammonium peroxodisulfate, can be employed, and, peroxides are desirable, and among them, ammonium peroxodisulfate is particularly preferable.
  • the usage of polymerization initiator is usually 0.1 to 3 % by weight for the total weight of total monomers.
  • the polymerization temperature is usually 40 to 120°C, preferably 50 to 70°C, and the polymerization time is 1 to 20 hours, preferably 0.5 to 5 hours.
  • the solvent to be used here includes water along with the above specific examples of the reaction solvent at the reaction of the (meth) acrylic acid derivative shown by the general formula [3] with the carboxylic acid compound shown by the general formula [4] or the salt thereof, and, water is preferable.
  • the amount of solvent to be used is usually 30 to 360 mL for 20 g of the total weight of polymer.
  • Content of the monomer unit shown by the general formula [1] in said copolymer is usually 50 % by mole or more and less than 100 % by mole, and 50 to 95 % by mole is preferable, and 50 to 60 % by mole is more preferable.
  • the content of the monomer unit shown by the general formula [2] in said copolymer is usually more than 0 % by mole and 50 % by mole or less, preferably it is 5 to 50 % by mole, and more preferably it is 40 to 50 % by mole.
  • monomer unit shown by the general formula [1] includes the same one as described in the paragraph of homopolymer which has a monomer unit shown by the general formula [1] in the above (1), and preferable one is also the same.
  • R 4 in the general formula [2] represents a hydrogen atom or a methyl group, and, a hydrogen atom is preferable.
  • "k" in the general formula [2] is usually 1 to 10, and, 4 to 8 is preferable, and 6 to 8 is more preferable.
  • the monomer unit shown by the general formula [2] includes, for example, the general formula [2-1] and [2-2], and, the general formula [2-1] is preferable.
  • k is the same as describe above
  • k is the same as describe above
  • Combination of the monomer unit shown by the general formula [1] and the monomer unit shown by the general formula [2] includes, for example, Polymer No. Monomer unit shown by general formula [1] Monomer unit shown by general formula [2] P-1 General formula [1-1] General formula [2-1] P-2 General formula [1-2] General formula [2-1] P-3 General formula [1-3] General formula [2-1] P-4 General formula [1-4] General formula [2-1] P-5 General formula [1-5] General formula [2-1] P-6 General formula [1-6] General formula [2-1] P-7 General formula [1-1] General formula [2-2] P-8 General formula [1-2] General formula [2-2] P-9 General formula [1-3] General formula [2-2] P-10 General formula [1-4] General formula [2-2] P-11 General formula [1-5] General formula [2-2] P-12 General formula [1-6] General formula [2-2] , and preferable combinations are Polymer No.
  • Monomer unit shown by general formula [1] Monomer unit shown by general formula [2] P-1 General formula [1-1] General formula [2-1] P-3 General formula [1-3] General formula [2-1] P-4 General formula [1-4] General formula [2-1] , among them, the combination whose monomer unit shown by the general formula [1] is the one which is shown by [1-1-1], [1-1-2], [1-3-1] or [1-4-1], and whose monomer unit shown by the general formula [2] is [2-1-1], is more preferable, and the combination whose monomer unit shown by the general formula [1] is [1-1-1], and whose monomer unit shown by the general formula [2] is [2-1-1] is particularly preferable.
  • a copolymer which has a monomer unit shown by the general formula [1] and a monomer unit shown by the general formula [2] for example, it can be produced by polymerizing a monomer shown by the following general formula [1'], (Wherein R 1 , R 2 , R 3 , X, n and m are the same as described above.), and a vinyl compound shown by the general formula [2'] (Wherein R 4 and k are the same as described above) by polymerization reaction according to a method well known per se, for example, a method described in JP-A-52-27713 .
  • the monomer shown by the general formula [1'] include the one which is in conformity to the monomer unit shown by the above-described general formula [1].
  • the monomer shown by the general formula [1'] can be obtained by the reaction of the (meth) acrylic acid derivative shown by the general formula [3] with the carboxylic acid compound shown by the general formula [4] or the salt thereof described in the paragraph of the method for production of a homopolymer which has monomer unit shown by the general formula [1].
  • vinyl compound shown by the general formula [2'] includes, for example, 4-pentenoic acid, 5-hexenoic acid, 6-heptenoic acid, 7-octenoic acid, 8-nonenoic acid, 9-decenoic acid, 10-undecenoic acid, 4-methyl-4-pentenoic acid, 5-methyl-5-hexenoic acid, 6-methyl-6-heptenoic acid, 7-methyl-7-octenoic acid, 8-methyl-8-nonenoic acid, 9-methyl-9-decenoic acid, 10-methyl-10-undecenoic acid etc., and, 7-octenoic acid, 8-nonenoic acid, 9-decenoic acid, 10-undecenoic acid etc. are preferable, and among them, 10-undecenoic acid is preferable especially.
  • the usage of the vinyl compound shown by the above-described general formula [2'] is usually 1 to 100 % by mole, preferably 3 to 100 % by mole, more preferably 50 to 100 % by mole for the amount of the monomer shown by the general formula [1'].
  • a conditionof the polymerization reaction of the monomer shown by the above-described general formula [1'] with the vinyl compound shown by the general formula [2'] includes the same one as polymerization reaction in the method for production of a polymer which has a monomer unit shown by the above-described general formula [1], and preferable one is also the same.
  • the method for production of a copolymer which has a monomer unit shown by the general formula [1] and a monomer unit shown by the general formula [2] is specifically performed as mentioned below.
  • One mole of the monomer shown by the general formula [1'] and 0.01 to 1 mole of the vinyl compound shown by the above-described general formula [2'] are dissolved in a 50 to 100 mL of mixed solution of water and DMF (1:2 to 2:1 as volume ratios), and followed by adding to the solution 0.1 to 3 % by weight of peroxide for the total weights of the above-described monomer and vinyl compound.
  • a polymerization reaction is carried out under argon atmosphere at 50 to 70°C for 1 to 20 hours.
  • the agglutination enhancer of the present invention comprises a polymer which has a monomer unit shown by the general formula [1] as described above, and more specifically, it comprises a homopolymer which has a monomer unit shown by the above-described general formula [1] or a copolymer which has a monomer unit shown by the above-described general formula [1] and a monomer unit shown by the general formula [2].
  • Said agglutination enhancer is preferably used by being dissolved in a reagent which is employed in the turbidimetric immunoassay among the immunoagglutination measurement methods, and among them, it is particularly preferable to be used by being dissolved in a reagent for the latex agglutination method which employs the latex as a carrier.
  • the above-described agglutination enhancer of the present invention is used by adding it so that its concentration in the reaction solution is usually 0.1 to 8 w/v%, preferably 0.1 to 4 w/v%, more preferably 0.1 to 2 w/v%.
  • said concentration is preferably changed its setting according to the analyte, and for example, in the case where the analyte is CRP and Fer, the agglutination enhancer is used by adding it so that its concentration in the reaction solution is usually 0.1 to 8 w/v%, preferably 0.1 to 4 w/v%, more preferably 0.1 to 1 w/v%.
  • the agglutination enhancer is used by adding it so that its concentration in the reaction solution is usually 0.1 to 7 w/v%, preferably 0.1 to 4 w/v%, more preferably 0.1 to 2 w/v%.
  • the immunoagglutination measurement method of the present invention may be performed according to the method of operation well known per se using various kinds of reagents which are used in the immunoagglutination measurement method well known per se (such as turbidimetric immunoassay, nephelometric immunoassay) which perform the measurement of analyte based on the agglutination etc. derived from the antigen-antibody reaction.
  • various kinds of reagents which are used in the immunoagglutination measurement method well known per se (such as turbidimetric immunoassay, nephelometric immunoassay) which perform the measurement of analyte based on the agglutination etc. derived from the antigen-antibody reaction.
  • it may be performed by carrying out the antigen-antibody reaction in the coexistence of the above-described agglutination enhancer of the present invention by bringing an antibody against the analyte or an antigen for the analyte contact with the analyte. More specifically, it may be performed by carrying out the antigen-antibody reaction in the coexistence of the above-described agglutination enhancer of the present invention by bringing an antibody against the analyte or a carrier supporting the antibody, or by bringing an antigen against the analyte or a carrier supporting the antigen into contact with the analyte.
  • the agglutination enhancer of the present invention in the above-described method may be coexisted at the above-described concentration in the reaction solution.
  • a specific method except for coexistence of the above-described agglutination enhancer of the present invention for example, when a method for measuring scattered light (nephelomety) is used, it may be carried out, for example, according to the method described in " Manual of Clinical Laboratory Medicine", 30th edition, 2nd printing, p.851-853 (1993), Kanehara & Co., Ltd.
  • a method for measuring transmitted light (turbidimetric immunoassay)
  • it may be carried out similarly according to the method described in the "Manual of Clinical Laboratory Medicine", 30th edition, 2nd printing, p.853-854 (1993), Kanehara & Co., Ltd. , etc.
  • the latex agglutination method in which the degree of agglutination of sensitized latex to an antibody against the analyte or an antigen for the analyte is measured based on the variation of the scattered light or transmitted light, and based on the results, measurement of the analyte is performed, it may be carried out, for example, according to the method described in " New Case Example of Utilization of Immunoassay Method and Application to Diagnostic Reagent ⁇ Development of Therapeutic Agent" (KEIEI KYOUIKU SYUPPANSYA) p. 103-187 , etc.
  • the buffer to be used at the reaction of the immunoagglutination measurement method of the present invention includes all the buffers usually to be used for the turbidimetric immunoassay and nephelometric immunoassay, such as, specifically, for example, Tris buffer, phosphate buffer, Veronal buffer, borate buffer and Good's buffer, and pH at the measurement reaction is not limited specifically as long as it is in a range where the antigen-antibody reaction is not restrained. It is usually selected suitably from a range between pH 6 and pH 10.
  • the measurement in the immunoagglutination measurement method of the present invention is performed by measuring scattered light or transmitted light, and the measurement may be carried out with the use of biochemical general-purpose equipment such as automated analyzer, spectrophotometer, and dedicated equipment for nephelometry measurement such as laser nephelometer.
  • biochemical general-purpose equipment such as automated analyzer, spectrophotometer, and dedicated equipment for nephelometry measurement such as laser nephelometer.
  • the measurement object component which can be measured by the immunoagglutination measurement method of the present invention, may be the one which can be measured by utilizing the antigen-antibody reaction, and it includes, for example, C-reactive protein (CRP), immunoglobulin G (IgG), immunoglobulin A (IgA), immunoglobulin M (IgM), immunoglobulin E (IgE), ASO (anti-streptolysin O), albumin, microalbumin, prealbumin, complement3 (C3), complement4 (C4), transferrin, haptoglobin, lipoprotein (a) (LP(a)), apolipoprotein A-I (ApoAI), apolipoprotein A-II (ApoAII), apolipoprotein B (ApoB), apolipoprotein C-II (ApoCII), apolipoprotein C-III (ApoCIII), apolipoprotein E (ApoE), rheumato
  • an agglutination enhancer comprising a copolymer which has a monomer unit shown by the general formula [1] and a monomer unit shown by the following general formula [2].
  • the reagents for the immunoagglutination measurement method of the present invention may be the one which comprises the above-described agglutination enhancer of the present invention, ant its content is usually 0.1 to 10 w/v %, preferably 0.1 to 5 w/v %, and more preferably 0.1 to 2 w/v% as a concentration in the reagent.
  • said concentration is preferable to change according to the analyte, and for example, in the case where the analyte is CRP and Fer, it is usually 0.1 to 10 w/v%, preferably 0.1 to 5 w/v%, more preferably 0.1 to 1 w/v%, and for example, in the case where the analyte is PSA, it is usually 0.1 to 10 w/v%, preferably 0.1 to 5 w/v%, more preferably 0.1 to 2 w/v%.
  • analyte in said reagent, beyond that, for example, in the case where the analyte is an antigen, it may comprise an antibody or an appropriate carrier (for example, latex and the like) supporting said antibody, or in the case where the analyte is an antibody, it may comprise an antigen or an appropriate carrier (for example, latex and the like) supporting said antigen.
  • an antibody or an appropriate carrier for example, latex and the like
  • an antigen or an appropriate carrier for example, latex and the like
  • said reaction regent may have buffer (for example, Tris buffer, phosphate buffer, Veronal buffer, borate buffer, Good's buffer, etc.), stabilizing agent (for example, albumin, globulin, water-soluble gelatin, surface active agent, carbohydrates, etc.), preservatives (for example, salicylic acid, benzoic acid, sodium azide, etc.), and other substances which are usually used in this field and do not disturb stability of coexisting reagent, nor inhibit antigen-antibody reaction.
  • concentration thereof may also be used within the range usually used in this field.
  • the measuring object in the reagent for immunoagglutination measurement method of the present invention includes the same one as described in the paragraph of the immunoagglutination measurement method of the present invention, and preferable one is also the same.
  • N-[2-(dimethylamino) ethyl] methacrylate (475 g, 3.0 moles, produced by Wako Pure Chemical Industries Ltd.) was added to said suspension, and then reaction was carried out under agitation and under reflux for 12 hours. After completion of the reaction, insoluble material was separated by filtration and washed with isopropanol to recover the wash solution. After the filtrate and the wash solution were combined and dried under reduced pressure, acetone was added to the obtained residue to precipitate crystals.
  • N-(4-dimethylamino) butanol (23 g, 0.2 moles, produced by Wako Pure Chemical Industries Ltd.) was dissolved in 200 mL of chloroform, and methacrylic acid chloride (25 g, 0.24 moles, produced by Wako Pure Chemical Industries Ltd.) was added to this solution under cooling on ice, and then reacted while agitating at room temperature for 1.5 hours. Subsequently, the obtained reaction solution was washed with saturated aqueous sodium bicarbonate and saturated aqueous sodium chloride, and then dried over magnesium sulfate. After that, the magnesium sulfate was removed by filtration, and the obtained filtrate was concentrated by reduced pressure to obtain 38 g of N-(4-dimethylamino) butyl methacrylate.
  • Potassium hydroxide (12 g, 0.21 moles) was dissolved in 150 mL of ethanol, and chloroacetic acid (20 g, 0.21 moles, produced by Wako Pure Chemical Industries Ltd.) was added, and then reacted under agitation at room temperature for 2 hours. Subsequently, crystals generated by the reaction were recovered by filtration, and washed with ethanol. After that, the washed crystals were suspended in 50 mL of ethanol, and N-(4-dimethylamino) butyl methacrylate (38 g, 0.2 moles) obtained in the above-described (1) was added to the suspension, and then, reacted under agitation and under reflux for 12 hours.
  • Monomer B (25 g) obtained in the above-described (2) was dissolved in 90 mL of ion-exchanged water, and then argon gas substitution was carried out for 30 minutes.
  • Ammonium peroxodisulfate solution (2 mL, 10 %) was added to this solution, and then reacted under agitation at 50 °C for 2 hours.
  • the reaction solution was purified using a dialysis tube [Spectra/Por 2 (molecular weight cut off: 12K to 14K, produced by Spectrum Laboratories, Inc.), ion-exchanged water; 5 L x 3 times]. After purification, the obtained polymer solution was lyophilized, and polymer 4 (17.2 g) was obtained.
  • Physical property of the polymer 4 was examined by way of GPC (Shodex SB-806M-HQ, produced by SHOWA DENKO K.K.). As a result, it was determined that the weight-average molecular weight was 807,920, and the molecular weight distribution was 3.895.
  • Monomer C (46 g) obtained in the above-described (1) was dissolved in 90 mL of ion-exchanged water, and then argon gas substitution was carried out for 30 minutes. Subsequently, 2 mL of 10 % ammonium peroxodisulfate solution was added to this solution, and then reacted under agitation at 50 °C for 2 hours. After completion of the reaction, the reaction solution was purified using a dialysis tube [Spectra/Por 2 (molecular weight cut off: 12K to 14K, produced by Spectrum Laboratories, Inc.), ion-exchanged water; 5 L x 3 times]. After purification, the obtained polymer solution was lyophilized, and polymer 5 (26.2 g) was obtained.
  • Physical property of the polymer 5 was examined by way of GPC (Shodex SB-806M-HQ, produced by SHOWA DENKO K.K.). As a result, it was determined that the weight-average molecular weight was 262,065, and the molecular weight distribution was 4.332.
  • Monomer D (21 g) obtained in the above-described (1) was dissolved in 90 mL of ion-exchanged water, and then argon gas substitution was carried out for 30 minutes. Subsequently, to this solution, 2 mL of 10 % ammonium peroxodisulfate solution was added, and then reacted under agitation at 50 °C for 2 hours. After completion of the reaction, the reaction solution was purified using a dialysis tube [Spectra/Por 2 (molecular weight cut off: 12K to 14K, produced by Spectrum Laboratories, Inc.), ion-exchanged water; 5 L x 3 times]. After the purification, the obtained polymer solution was lyophilized, and polymer 6 (13.5 g) was obtained.
  • Physical property of the polymer 6 was examined by way of GPC (Shodex SB-806M-HQ, produced by SHOWA DENKO K.K.). As a result, it was determined that the weight-average molecular weight was 407,359, and the molecular weight distribution was 2.375.
  • Monomer A (22 g) obtained in the above-described Synthetic Example 1 and 10-undecenoic acid (1.8 g, produced by Wako Pure Chemical Industries Ltd.) were dissolved in a mixed solvent of 50 mL of ion-exchanged water and 40 mL of DMF, and then argon gas substitution was carried out for 30 minutes. Subsequently, 2 mL of 10 % ammonium peroxodisulfate solution was added to this solution, and then reacted under agitation at 50 °C for 2 hours.
  • the reaction solution was purified using a dialysis tube [Spectra/Por 2 (molecular weight cut off: 12K to 14K, produced by Spectrum Laboratories, Inc.), ion-exchanged water; 5 L x 3 times]. After purification, the obtained polymer solution was lyophilized, and copolymer 1 (16.2 g) was obtained. In addition, the copolymer 1 comprises 40 % by mole of undecenoic acid derived monomer unit (hereinafter, abbreviated as monomer unit E).
  • monomer unit E undecenoic acid derived monomer unit
  • Physical property of the copolymer 1 was examined by way of GPC (Shodex SB-806M-HQ, produced by SHOWA DENKO K.K.). As a result, it was determined that the weight-average molecular weight was 658,206, and the molecular weight distribution was 2.216.
  • Monomer A (22 g) obtained in the above-described Synthetic Example 1 and 10-undecenoic acid (7.4 g, produced by Wako Pure Chemical Industries Ltd.) were dissolved in a mixed solvent of 50 mL of ion-exchanged water and 40 mL of DMF, and then argon gas substitution was carried out for 30 minutes. Subsequently, 2 mL of 10 % ammonium peroxodisulfate solution was added to this solution, and then reacted under agitation at 50 °C for 2 hours.
  • the reaction solution was purified using a dialysis tube [Spectra/Por 2 (molecular weight cut off: 12K to 14K, produced by Spectrum Laboratories, Inc.), ion-exchanged water; 5 L x 3 times]. After the purification, the obtained polymer solution was lyophilized, and copolymer 2 (19.4 g) was obtained. In addition, the copolymer 2 comprises 5 % by mole of monomer unit E.
  • Physical property of the copolymer 2 was examined by way of GPC (Shodex SB-806M-HQ, produced by SHOWA DENKO K.K.). As a result, it was determined that the weight-average molecular weight was 957,680, and the molecular weight distribution was 2.041.
  • Example 1 Measurement of CRP by latex immunoagglutination measurement method using-polymers with different molecular weight as an immunoagglutination enhancer
  • Borate buffer solution (4.5 mL, 50 mM, pH 7.5) containing 1 mg/mL of anti-human CRP goat polyclonal antibody (produced by Oriental Yeast Co., Ltd.) was mixed with 0.5 mL of 10 w/v% aqueous solution of polystyrene latex with 0.12 ⁇ m in particle size (produced by Nippon Paint Co., Ltd.), and reacted at 7 °C overnight. Then, 5 mL of the obtained suspension was mixed with borate buffer solution (5 mL, 50 mM, pH7.5) containing 2.5 w/v% bovine serum albumin (BSA), and reacted at 7 °C for 2 hours.
  • BSA bovine serum albumin
  • LT ⁇ CRP-HS Calibrator Set HO manufactured by Wako Pure Chemical Industries Ltd., and CRP concentration thereof is 0.2 mg/dL, 1.0 mg/dL, 4.0 mg/dL, 18.0 mg/dL, and 35.0 mg/dL, respectively.
  • Tris buffer solutions (0.1 M, pH 8.0), which contain 0.4 w/v% of polymer 1, polymer 2 or polymer 3 synthesized in Synthetic Example 1 as an agglutination enhancer, and include 0.1 % BSA and 1 % NaCl, were prepared, and thus three kinds of first reagents were prepared.
  • the anti-human CRP (12 mL) antibody-sensitized latex 1) and the anti-human CRP (8 mL) antibody-sensitized latex 2) which were prepared in the above-described (1) were mixed, and used as a second reagent.
  • the CRP concentration in a sample was measured using the above-described sample, the above-described first reagent and the above-described second reagent under the following condition:
  • Sample 7.5 ⁇ L (diluted five fold with saline);
  • First reagent 100 ⁇ L
  • Second reagent 25 ⁇ L
  • Measurement method 2 point-end method (34 to 65);
  • Dominant wavelength 596 nm.
  • the CRP was measured by the same method as described in Example 1 except for using 0.1 M Tris buffer solution (pH 8.0) which contains 0.4 w/v% of polyethylene glycol 6,000 (PEG 6,000, produced by Wako Pure Chemical Industries Ltd.) or MPC polymer (produced by NOF Corporation) instead of polymer 1 to 3 of Example 1 as an agglutination enhancer, and includes 0.1 % BSA and 1 % NaCl.
  • the results were shown in Table 1 in conjunction with the results of Example 1.
  • Example 2 Measurement of Fer by latex immunoagglutination measurement method using polymers with different molecular weight as an immunoagglutination enhancer
  • Borate buffer solution (1 mL, 50 mM, pH 7.5) containing 0.6 mg/mL of anti-human Fer rabbit polyclonal antibody (produced by Dako Corporation) was mixed with borate buffer solution (1 mL, 50 mM, pH 7.5) in which polystyrene latex with 0.3 ⁇ m in particle size (produced by Sekisui Chemical Co., Ltd.) was suspended so as to provide 2 w/v%, and reacted at 25 °C for 2 hours. Then, 2 mL of the obtained suspension was mixed with borate buffer solution (2 mL, 50 mM, pH7.5) containing 1.25 w/v% BSA, and reacted at 7 °C for 2 hours.
  • Comparative Example 2 Measurement of Fer by latex immunoagglutination measurement method using conventional immunoagglutination enhancer
  • the Fer was measured by the same method as described in Example 2 except for using HEPES buffer solution (0.1 M, pH 8.0) which contains 0.4 w/v% of polyethylene glycol 6,000 (PEG 6,000, produced by Wako Pure Chemical Industries Ltd.) or MPC polymer (produced by NOF Corporation) instead of polymer 1 to 3 of Example 2 as an agglutination enhancer, and includes 0.1 % BSA and 1 % NaCl.
  • HEPES buffer solution 0.1 M, pH 8.0
  • MPC polymer produced by NOF Corporation
  • Example 3 Measurement of PSA by latex immunoagglutination measurement method using polymers with different molecular weight as an immunoagglutination enhancer
  • Borate buffer solution (1 mL, 50 mM, pH 7.1) containing 0.6 mg/mL of anti-human PSA monoclonal antibody (Clone No. PSA10; produced by Wako Pure Chemical Industries Ltd.) was mixed with borate buffer solution (1 mL, 50 mM, pH 7.1) in which polystyrene latex with 0.28 ⁇ m in particle size (produced by Sekisui Chemical Co., Ltd.) was suspended so as to provide 2 w/v%, and reacted at 25 °C for 2 hours.
  • borate buffer solution (1 mL, 50 mM, pH7.1) containing 1.4 mg of anti-human PSA monoclonal antibody (Clone No. PSA14; produced by Wako Pure Chemical Industries Ltd.) was mixed with borate buffer solution (1 mL, 50 mM, pH 7.1) in which polystyrene latex with 0.15 ⁇ m in particle size (produced by Sekisui Chemical Co., Ltd.) was suspended so as to provide 2 w/v%, and used it as an anti-human PSA antibody-sensitized latex solution 2).
  • Phosphate buffer solution (10 mM phosphate buffer solution containing 1 w/v% BSA and 0.85 % NaCl) was used as a reagent blanktest sample.
  • PSA Calibrator Set (produced by Wako Pure Chemical Industries Ltd.; which includes those with PSA concentration, 5.0 ng/mL, 10.0 ng/mL, 39.8 ng/mL, 69.3 ng/mL, and 98.6 ng/mL, respectively) was used.
  • HEPES-NaOH buffer solutions (0.1 M, pH 7.0), which contain 0.75 w/v% of polymer 1, polymer 2 or polymer 3 synthesized in Synthetic Example 1 as an agglutination enhancer, and include 0.1 % BSA and 1 % NaCl, were prepared, and thus three kinds of first reagents were prepared.
  • the PSA concentration in a sample was measured using the above-described sample, above-described first reagent and above-described second reagent under the following condition:
  • the PSA was measured by the same method as described in Example 3 except for using HEPES-NaOH buffer solution (0.1 M, pH 8.0) which contains 0.4 w/v% of polyethylene glycol 6,000 (PEG 6,000, produced by Wako Pure Chemical Industries Ltd.) or MPC polymer (produced by NOF Corporation) instead of polymer 1 to 3 of Example 3 as an agglutination enhancer, and includes 0.1 % BSA and 1 % NaCl.
  • HEPES-NaOH buffer solution 0.1 M, pH 8.0
  • MPC polymer produced by NOF Corporation
  • Example 4 Measurement of CRP by latex immunoagglutination measurement method using various types of polymers as an immunoagglutination enhancer
  • the CRP was measured by the same method as described in Example 1 except for using 0.1 M Tris buffer solution (pH 8.0) which contains 0.4 w/v% of polymer 4 to 6 as an agglutination enhancer instead of polymer 1 to 3, and includes 0.1 % BSA and 1 % NaCl as a first reagent, and.
  • the results were shown in Table 4.
  • Example 5 Measurement of Fer by latex immunoagglutination measurement method using various types of polymers as an immunoagglutination enhancer
  • the Fer was measured by the same method as described in Example 2 except for using 0.1 M Tris buffer solution (pH 8.0) which contains 0.4 w/v% polymer 4 to 6 as an agglutination enhancer instead of polymer 1 to 3 and includes 0.1 % BSA and 1 % NaCl as a first reagent. The results were shown in Table 5.
  • Example 6 Measurement of PSA by latex immunoagglutination measurement method using various types of polymers as an immunoagglutination enhancer
  • the PSA was measured by the same method as described in Example 3 except for using 0.1 M HEPES-NaOH buffer solution (pH 7.0) which contains 0.4 w/v% polymer 4 to 6 instead of polymer 1 to 3 as an agglutination enhancer and includes 0.1 % BSA and 1 % NaCl as a first reagent. The results were shown in Table 6.
  • Example 7 Measurement of CRP by latex immunoagglutination measurement method using a copolymer as an immunoagglutination enhancer
  • the CRP was measured by the same method as described in Example 1 except for using 0.1 M Tris buffer solution (pH 8.0) which contains 0.4 w/v% of copolymer 1 or 2 as an agglutination enhancer instead of polymer 1 to 3 and includes 0.1 % BSA and 1 % NaCl as a first reagent.
  • the results were shown in Table 7.
  • Example 8 Measurement of Fer by latex immunoagglutination measurement method using a copolymer as an immunoagglutination enhancer
  • the Fer was measured by the same method as described in Example 2 except for using 0.1 M HEPES-NaOH buffer solution (pH 7.0) which contains 0.4 w/v% copolymer 1 or 2 as an agglutination enhancer instead of polymer 1 to 3 and includes 0.1 % BSA and 1 % NaCl as a first reagent. The results were shown in Table 8.
  • Example 9 Measurement of PSA by latex immunoagglutination measurement method using a copolymer as an immunoagglutination enhancer
  • Example 10 Measurement of CK-MB by latex immunoagglutination measurement method using a copolymer as an immunoagglutination enhancer
  • Borate buffer solution (1 mL, 50 mM, pH 7.5) containing 0.8 mg/mL of anti-human CK-MB monoclonal antibody (Clone MAK ⁇ CK-MB> M-7.4.5-IgG; produced by Roche Diagnostics GmbH) was mixed with borate buffer solution (1 mL, 50 mM, pH 7.5) in which polystyrene latex with 0.4 ⁇ m in particle size (produced by Fujikura Kasei Co., Ltd.) was suspended so as to contain 2 w/v%, and reacted at 25 °C for 2 hours.
  • borate buffer solution (1 mL, 50 mM, pH7.5) containing 0.8 mg of anti-human CK-MB monoclonal antibody (Clone MAK ⁇ CK-MB> M-6.12.47-IgG; produced by Roche Diagnostics GmbH) was mixed with borate buffer solution (1 mL, 50 mM, pH 7.5) in which polystyrene latex with 0.4 ⁇ m in particle size (produced by Fujikuraanalyte Kasei Co., Ltd.) was suspended so as to contain 2 w/v%, and used it as an anti-human CK-MB antibody-sensitized latex solution 2).
  • Saline (0.85 % NaCl) was used for a reagent blank test sample.
  • the sample was prepared by diluting CK-MB antigen (CK-MB of human origin, produced by Cliniqa Diagnostics Inc.) with phosphate buffer solution (containing 10 mM phosphate, 1 w/v% BSA and 0.85 % NaCl) so that its concentration is 5.2 ng/mL, 19.0 ng/mL, 47.9 ng/mL, 98.7 ng/mL, and 204.3 ng/mL, respectively, and used them as samples.
  • CK-MB antigen CK-MB of human origin, produced by Cliniqa Diagnostics Inc.
  • phosphate buffer solution containing 10 mM phosphate, 1 w/v% BSA and 0.85 % NaCl
  • HEPES-NaOH solutions (0.1 M, pH 7.0), which contains 0.75 w/v% of polymer 1, copolymer 1, or copolymer 2 and includes 0.1 % BSA and 1 % NaCl, were prepared, and thus three kinds of first reagents were prepared.
  • the anti-human CK-MB antibody-sensitized latex solution 1) and 2) (2 mL each) prepared in the above-described (1) were suspended in 50 mM borate buffer solution (pH7.5) containing 0.5 w/v% BSA and mixed, and used it as a second reagent.
  • CK-MB concentration in a sample was measured using the above-described sample, above-described first reagent and above-described second reagent under the following condition:
  • Comparative Example 4 Measurement of CK-MB by latex immunoagglutination measurement method using conventional immunoagglutination enhancer
  • the CK-MB was measured by the same method as described in Example 10 except for using 0.1 M HEPES-NaOH buffer solution (pH 7.0) which contains 0.75 w/v% polyethylene glycol 6,000 (PEG 6,000, produced by Wako Pure Chemical Industries Ltd.) or MPC polymer (produced by NOF Corporation) as an agglutination enhancer instead of polymer 1, copolymer 1 and copolymer 2 and includes 0.1 % BSA and 1 % NaCl as a first reagent.
  • the results were shown in Table 10 in conjunction with the results of Example 10.
  • Example 11 Measurement of CRP by latex immunoagglutination measurement method in different content of immunoagglutination enhancer
  • the CRP was measured by the same method as described in Example 1 except for using 0.1 M Tris buffer solution (pH 8.0) which contains 0.24 w/v%, 0.32 w/v%, 0.56 w/v%, or 0.72 w/v% of polymer 1 and includes 0.1 % BSA and 1 % NaCl as a first reagent. The results were shown in Table 11.
  • Example 12 Measurement of Fer by latex immunoagglutination measurement method in different content of immunoagglutination enhancer
  • the Fer was measured by the same method as described in Example 2 except for using 0.1 M HEPES-NaOH buffer solution (pH 7.0) which contains 0.25 w/v%, 0.31 w/v%, 0.49 w/v%, or 0.61 w/v% of polymer 1 and includes 0.1 % BSA and 1 % NaCl as a first reagent.
  • 0.1 M HEPES-NaOH buffer solution pH 7.0
  • Example 13 Measurement of PSA by latex immunoagglutination measurement method in different content of immunoagglutination enhancer
  • the PSA was measured by the same method as described in Example 3 except for using 0.1 M HEPES-NaOH buffer solution (pH 7.0) which contains 0.60 w/v%, 0.90 w/v%, 1.35 w/v%, or 1.50 w/v% of polymer 1 and includes 0.1 % BSA and 1 % NaCl as a first reagent.
  • 0.1 M HEPES-NaOH buffer solution pH 7.0

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Claims (11)

  1. Utilisation d'un polymère en tant qu'agent renforçant l'agglutination pour un procédé de mesure de l'immunoagglutination, le polymère ayant un motif de monomère illustré par la formule générale [1] suivante :
    Figure imgb0047
     dans laquelle R1 représente un atome d'hydrogène ou un groupe méthyle ; R2 et R3 représentent indépendamment un groupe méthyle ou un groupe éthyle, respectivement ; X représente un groupe -NH- ou un atome d'oxygène ; n représente un nombre entier de 1 à 6 ; et m représente un nombre entier de 1 à 3.
  2. Utilisation selon la revendication 1, dans laquelle le poids moléculaire moyen en poids du polymère est de 50 000 à 3 000 000.
  3. Utilisation selon la revendication 1, dans laquelle R2 et R3 dans la formule générale [1] sont tous deux un groupe méthyle.
  4. Utilisation selon la revendication 1, dans laquelle n vaut de 2 à 4 et m vaut 1 dans la formule générale [1].
  5. Agent renforçant l'agglutination pour un procédé de mesure de l'immunoagglutination qui comprend un copolymère ayant un motif de monomère illustré par la formule générale [1] suivante :
    Figure imgb0048
     dans laquelle R1 représente un atome d'hydrogène ou un groupe méthyle ; R2 et R3 représentent indépendamment un groupe méthyle ou un groupe éthyle, respectivement ; X représente un groupe -NH- ou un atome d'oxygène ; n représente un nombre entier de 1 à 6 ; et m représente un nombre entier de 1 à 3 ; et
    un motif de monomère illustré par la formule générale [2] suivante :
    Figure imgb0049
     dans laquelle R4 représente un atome d'hydrogène ou un groupe méthyle, et k représente un nombre entier de 1 à 10.
  6. Agent renforçant l'agglutination selon la revendication 5, dans laquelle la teneur du motif de monomère illustré par la formule générale [1] dans le copolymère est de 50 % en mole ou plus et inférieure à 100 % en mole.
  7. Agent renforçant l'agglutination selon la revendication 5, dans laquelle R4 est un atome d'hydrogène et k vaut de 4 à 8 dans la formule générale [2].
  8. Réactif pour un procédé de mesure de l'immunoagglutination qui comprend l'agent renforçant l'agglutination selon la revendication 5.
  9. Réactif pour un procédé de mesure de l'immunoagglutination selon la revendication 8, lequel réactif est destiné à une utilisation pour la mesure d'une protéine réactive C (CRP), de la ferritine sérique (Fer), de l'antigène spécifique de la prostate (PSA) ou de la créatine kinase-MB (CK-MB).
  10. Procédé de mesure de l'immunoagglutination qui comprend le fait qu'en la coexistence d'un agent renforçant l'agglutination comprenant un polymère ayant un motif de monomère illustré par la formule générale [1] suivante, un anticorps ou un antigène contre un analyte est amené en contact avec l'analyte pour déterminer la présence ou la concentration de l'analyte dans un échantillon biologique,
    Figure imgb0050
     dans laquelle R1 représente un atome d'hydrogène ou un groupe méthyle ; R2 et R3 représentent indépendamment un groupe méthyle ou un groupe éthyle, respectivement ; X représente un groupe -NH- ou un atome d'oxygène ; n représente un nombre entier de 1 à 6 ; et m représente un nombre entier de 1 à 3.
  11. Procédé de mesure de l'immunoagglutination selon la revendication 10, dans lequel l'analyte est destiné à une utilisation dans la mesure de la protéine réactive C (CRP), de la ferritine sérique (Fer), de l'antigène spécifique de la prostate (PSA) ou de la créatine kinase-MB (CK-MB).
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